Mid-cell localization of the cell division septum in bacteria such as E. coli is controlled by a set of proteins including MinC, MinD, MinE, and FtsZ. FtsZ is the first structural component of the septum to polymerize on the inner membrane at the mid-cell when the cell starts to divide. FtsZ polymerization is limited to mid-cell by the action of the three Min proteins. MinC is an inhibitor of FtsZ polymerization, but on its own, it does not exhibit specific membrane localization. Instead, it binds to MinD dimers that form in the presence of ATP and binds membrane: MinD is an ATP-dependent membrane binding protein. The two proteins generally co-localize on the membrane. MinE also interacts with MinD dimer and can displace MinC from MinD. More importantly, MinE controls MinD ATPase activity and also influences its membrane interaction, and hence its membrane association/dissociation dynamics. In vivo imaging studies have demonstrated the oscillating pattern formation by MinD and MinE proteins, resulting in a concentration minimum of MinD, and hence MinC, at the mid-cell region when averaged over time. This observation explained why FtsZ polymerization is restricted to mid-cell. However, a detailed molecular mechanism of this bio-patterning reaction system is still poorly understood. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of this reaction system by combining a variety of techniques, including a reconstituted cell-free reaction system we have established that recapitulates aspects of in vivo system dynamics. Techniques and instruments have been developed to study these dynamic reaction systems in vitro by using a sensitive fluorescence microscope system. By using fluorescence-labeled MinD and MinE proteins, assembly and disassembly of these proteins on a supported lipid bilayer on the slide glass surface are monitored under a variety of reaction conditions. We successfully reconstituted a variety of modes of self-organized dynamic pattern formation by MinD and MinE proteins in the presence of ATP on the membrane surface. We also study the kinetic parameters of the biochemical reaction steps involved in the reaction. Recent progress allowed us to propose the first comprehensive detailed molecular mechanistic model for this reaction system that is supported by a body of experimental observations we have accumulated. Further mechanistic details of the dynamic pattern organization are currently studied combining biochemical, biophysical and mathematical approaches. Currently, conformational dynamics of MinE protein that plays critical roles in the control of MinD-DNA interaction dynamics is investigated using NMR techniques. In addition, we are investigating the nature of MinD-MinE protein complexes that form on the membrane as transient reaction intermediates in order to further refine our detailed molecular mechanistic model. This study is in part aimed at advancement of our knowledge on how a set of protein molecules could orchestrate a spatial control of cellular events that occur with a much larger length-scale than the individual protein molecules involved, without assembling polymeric protein filaments that spans the distance.